Céline DerMardirossian

A novel and specific NADPH oxidase-1 (Nox1) small-molecule inhibitor blocks the formation of functional invadopodia in human colon cancer cells

The NADPH oxidase (Nox) proteins catalyze the regulated formation of reactive oxygen species (ROS), which play key roles as signaling molecules in several physiological and pathophysiological processes. ROS generation by the Nox1 member of the Nox family is necessary for the formation of extracellular matrix (ECM)-degrading, actin-rich cellular structures known as invadopodia. Selective inhibition of Nox isoforms can provide reversible, mechanistic insights into these cellular processes in contrast to scavenging or inhibition of ROS production. Currently no specific Nox inhibitors have been described. Here, by high-throughput screening, we identify a subset of phenothiazines, 2-acetylphenothiazine (here referred to as ML171) (and its related 2-(trifluoromethyl)-phenothiazine) as nanomolar, cell-active, and specific Nox1 inhibitors that potently block Nox1-dependent ROS generation, with only marginal activity on other cellular ROS-producing enzymes and receptors including the other Nox isoforms. ML171 also blocks the ROS-dependent formation of ECM-degrading invadopodia in colon cancer cells. Such effects can be reversed by overexpression of Nox1 protein, which is suggestive of a selective mechanism of inhibition of Nox1 by this compound. These results elucidate the relevance of Nox1-dependent ROS generation in mechanisms of cancer invasion and define ML171 as a useful Nox1 chemical probe and potential therapeutic agent for inhibition of cancer cell invasion.

Regulation of hypoxic neuronal death signaling by neuroglobin

The signal transduction pathways in volved in neuronal death are not well understood. Neuroglobin (Ngb), a recently discovered vertebrate globin expressed predominantly in the brain, shows increased expression in neurons in response to oxygen deprivation and protects neurons from ischemic and hypoxic death. The mechanism of this neuroprotection is unclear. We examined the surface distribution of raft membrane microdomains in cortical neuron cultures during hypoxia using the raft marker cholera toxin B (CTx‐B) subunit. Mechanistically, we demonstrate that hypoxia induces rapid polarization of somal mem branes and aggregation of microdomains with the sub jacent mitochondrial network. This signaling complex is formed well before neurons commit to die, consis tent with an early role in death signal transduction. Neurons from Ngb‐overexpressing transgenic (Ngb‐Tg) mice do not undergo microdomain polarization or mitochondrial aggregation in response to, and are resistant to death from hypoxia. We link the protective actions of Ngb to inhibition of Pak1 kinase activity and Racl‐GDP‐dissociation inhibitor disassociation, and in hibition of actin assembly and death‐signaling module polarization.— Khan, A. A., Mao, X. O., Banwait, S., DerMardirossian, C. M., Bokoch, G. M., Jin, K., Greenberg, D. A. Regulation of hypoxic neuronal death signaling by neuroglobin. FASEB J. 22, 1737–1747 (2008)

Pak1 regulates focal adhesion strength, myosin IIA distribution, and actin dynamics to optimize cell migration

p21-activated kinases are essential for spatial and temporal coordination of cytoskeletal dynamics with cellular adhesion during cell migration.

c-Src–Mediated Phosphorylation of NoxA1 and Tks4 Induces the Reactive Oxygen Species (ROS)–Dependent Formation of Functional Invadopodia in Human Colon Cancer Cells

Reactive oxygen species (ROS) generated by the NADPH oxidase system have been shown to be necessary for the invadopodia formation and function. We show here that the abolishment of Src-mediated phosphorylation of NoxA1 and Tks4 blocks their binding, decreases Nox1-dependent ROS generation, and inhibits the invadopodia formation and ECM degradation.

The Microtubule-associated Rho Activating Factor GEF-H1 interacts with Exocyst complex to regulate Vesicle Traffic

The exocyst complex plays a critical role in targeting and tethering vesicles to specific sites of the plasma membrane. These events are crucial for polarized delivery of membrane components to the cell surface, which is critical for cell motility and division. Though Rho GTPases are involved in regulating actin dynamics and membrane trafficking, their role in exocyst-mediated vesicle targeting is not very clear. Herein, we present evidence that depletion of GEF-H1, a guanine nucleotide exchange factor for Rho proteins, affects vesicle trafficking. Interestingly, we found that GEF-H1 directly binds to exocyst component Sec5 in a Ral GTPase-dependent manner. This interaction promotes RhoA activation, which then regulates exocyst assembly/localization and exocytosis. Taken together, our work defines a mechanism for RhoA activation in response to RalA-Sec5 signaling and involvement of GEF-H1/RhoA pathway in the regulation of vesicle trafficking.

Direct interaction between Tks proteins and the N-terminal proline-rich region (PRR) of NoxA1 mediates Nox1-dependent ROS generation

NADPH oxidase (Nox) family enzymes are one of the main sources of cellular reactive oxygen species (ROS), which have been implicated in several physiological and pathophysiological processes. To date seven members of this family have been reported, including Nox1-5 and Duox1 and 2. With the exception of Nox2, the regulation of the Nox enzymes is still poorly understood. Nox1 is highly expressed in the colon, and requires two cytosolic regulators, the organizer subunit NoxO1 and the activator subunit NoxA1, as well as the binding of Rac1 GTPase, for its activity. Recently, we identified the c-Src substrate proteins Tks4 and Tks5 as functional members of a p47(phox)-related organizer superfamily. As a functional consequence of this interaction, Nox1 localizes to invadopodia, actin-rich membrane protrusions of cancer cells which facilitate pericellular proteolysis and invasive behavior. Here, we report that Tks4 and Tks5 directly bind to NoxA1. Moreover, the integrity of the N-terminal PRR of NoxA1 is essential for this direct interaction with the Tks proteins. When the PRR in NoxA1 is disrupted, Tks proteins cannot bind NoxA1 and lose their ability to support Nox1-dependent ROS generation. Consistent with this, Tks4 and Tks5 are unable to act as organizers for Nox2 because of their inability to interact with p67(phox), which lacks the N-terminal PRR, thus conferring a unique specificity to Tks4 and 5. Taken together, these results clarify the molecular basis for the interaction between NoxA1 and the Tks proteins and may provide new insights into the pharmacological design of a more effective anti-metastatic strategy.

Kinome-wide Functional Analysis Highlights the Role of Cytoskeletal Remodeling in Somatic Cell Reprogramming

The creation of induced pluripotent stem cells (iPSCs) from somatic cells by ectopic expression of transcription factors has galvanized the fields of regenerative medicine and developmental biology. Here, we report a kinome-wide RNAi-based analysis to identify kinases that regulate somatic cell reprogramming to iPSCs. We prepared 3,686 small hairpin RNA (shRNA) lentiviruses targeting 734 kinase genes covering the entire mouse kinome and individually examined their effects on iPSC generation. We identified 59 kinases as barriers to iPSC generation and characterized seven of them further. We found that shRNA-mediated knockdown of the serine/threonine kinases TESK1 or LIMK2 promoted mesenchymal-to-epithelial transition, decreased COFILIN phosphorylation, and disrupted Actin filament structures during reprogramming of mouse embryonic fibroblasts. Similarly, knockdown of TESK1 in human fibroblasts also promoted reprogramming to iPSCs. Our study reveals the breadth of kinase networks regulating pluripotency and identifies a role for cytoskeletal remodeling in modulating the somatic cell reprogramming process.

Reactive Oxygen Species Regulate Protrusion Efficiency by Controlling Actin Dynamics

Productive protrusions allowing motile cells to sense and migrate toward a chemotactic gradient of reactive oxygen species (ROS) require a tight control of the actin cytoskeleton. However, the mechanisms of how ROS affect cell protrusion and actin dynamics are not well elucidated yet. We show here that ROS induce the formation of a persistent protrusion. In migrating epithelial cells, protrusion of the leading edge requires the precise regulation of the lamellipodium and lamella F-actin networks. Using fluorescent speckle microscopy, we showed that, upon ROS stimulation, the F-actin retrograde flow is enhanced in the lamellipodium. This event coincides with an increase of cofilin activity, free barbed ends formation, Arp2/3 recruitment, and ERK activity at the cell edge. In addition, we observed an acceleration of the F-actin flow in the lamella of ROS-stimulated cells, which correlates with an enhancement of the cell contractility. Thus, this study demonstrates that ROS modulate both the lamellipodium and the lamella networks to control protrusion efficiency.

Protein tyrosine phosphatase-PEST and β8 integrin regulate spatiotemporal patterns of RhoGDI1 activation in migrating cells.

ABSTRACT Directional cell motility is essential for normal development and physiology, although how motile cells spatiotemporally activate signaling events remains largely unknown. Here, we have characterized an adhesion and signaling unit comprised of protein tyrosine phosphatase (PTP)-PEST and the extracellular matrix (ECM) adhesion receptor β8 integrin that plays essential roles in directional cell motility. β8 integrin and PTP-PEST form protein complexes at the leading edge of migrating cells and balance patterns of Rac1 and Cdc42 signaling by controlling the subcellular localization and phosphorylation status of Rho GDP dissociation inhibitor 1 (RhoGDI1). Translocation of Src-phosphorylated RhoGDI1 to the cells leading edge promotes local activation of Rac1 and Cdc42, whereas dephosphorylation of RhoGDI1 by integrin-bound PTP-PEST promotes RhoGDI1 release from the membrane and sequestration of inactive Rac1/Cdc42 in the cytoplasm. Collectively, these data reveal a finely tuned regulatory mechanism for controlling signaling events at the leading edge of directionally migrating cells.

Affinity-based assay of Rho guanosine triphosphatase activation.

The recognition that Rho guanosine triphosphatases (GTPases) (Rho, Rac, and Cdc42) play important regulatory roles in many areas of cell biology has made the ability to measure their activity in cells an important biological tool. Because Rho GTPases become activated by conversion from guanosine diphosphate-bound states to guanosine triphosphate (GTP)-bound forms, affinity-based methods to detect the formation of GTP-Rho GTPases have been developed and are widely used for the purpose of assessing Rho GTPase activities in biological studies.